The aim of the proposed research is to examine the role of Ca2+ as the transmitter signal in gating Ca2+ release channels of the SR (Ryanodine receptors) and to evaluate the role of phospholamban in regulation of relaxation in mammalian cardiomyocytes. Central to the question of Ca2+ gating is the role and effectiveness of various sarcolemmal Ca2+ transporters in gating the Ca2+ release from the SR. WE shall, therefore, first evaluate the gating efficiency of Ca2+ delivered by the Ca2+ channel versus that transported by the Na+-Ca2+ exchanger and the Ca2+-selective Na+ channel (transformed by Atrionatriuretic Peptide in atrial and ventricular myocytes of mammalian heart by quantifying the integral of Ca2+ charge traversing the sarcolemma sufficient to trigger Ca2+ release. The hypothesis at the core of this study is that there is privileged communication between the Ca2+ channels (DHP receptors) and the Ryanodine receptors. The "privileged access concept" will be further probed by testing the accessibility of Ryanodine receptor blockers (ruthenium red and caged Mg2+) and caged Ca2+-buffers (DM-Nitrophen and Diazo-2) to the Ca2+-sensing site of the SR release channels in intact myocytes. The implications of the limited access hypothesis on the inactivation of the Ryanodine receptor by Ca2+ will be explored by a novel "epi-axial" method to photorelease Ca2+ in sub-sarcolemmal space. The level of expression of various sarcolemmal Ca2+-transporting proteins in cardiac myocytes is critical to this evaluation, requiring quantification of gating efficiency of different Ca2+ delivery systems in other mammalian species. Thus, the effectiveness of Ca2+ signalling via the Na+-Ca2+ exchanger, compared to the Ca2+ channel, will be probed in hamster myocytes, which show a ten-fold higher exchanger current density than the rat myocytes. Two pathological and one development model of E-C coupling will be examined which represent up-regulated expression of either the exchanger (myopathic hamster) or Ca2+ channels (spontaneously hypertensive rats), and the low density of SR release channels (neonatal human and cat cardiomyocytes). Detailed examination of Ca2+ release and uptake in AT-1 atrial tumor cell line will be carried out, not only because AT-1 cells do not express phospholamban (Ca2+ pump regulatory protein), but also because these cells are good candidates for genetic manipulation of various molecular components of E-C coupling. Probing the role of phospholamban in regulation of Ca2+ uptake will include evaluation of the kinetics of phosphorylation of phospholamban by injection of 2D12 antibody and photorelease of caged cAMP vs. alterations in the Ca2+ sensitivity of the myofilaments in regulating the relaxant effects of isoproterenol in AT-1 cells and normal ventricular myocytes. Studies on the regulation of Ca2+ uptake will be carried out in AT-1 cells transfected with various fragments of phospholamban to pinpoint its structure-function relationship. A newly-developed single-cell spectrofluorometer will be used in some experiments to monitor the activity of more than one dye simultaneously and quantify the photorelease of caged Ca2+ prior to and following photorelease. By exploring the properties of Ca2+ release and uptake systems in their native environment, we shall, to some extent, follow the signalling pathways to their points of origin and characterize a) the adequacy of our experimental interventions, b) the role of the Ca2+ channel and Na+-Ca2+ exchanger and their differential expression in different animals and pathological states, and c) the regulation of the Ca2+-ATPase by phospholamban.